The experiment was carried Measuring activity of enzyme like exoglucanase, endoglucanase and xylanase and the nutritional quality of different strains of milky mushroom namely viz., CI-1, CI-2, CI-3, MG-4, CI-5 and APK-2 in the study. Among the five strains tested, CI-3 recorded maximum activity of endoglucanase, exoglucanase, and endoxylanase (1.50, 1.02, 1.66 μ moles sugar/min/mg protein,) followed by strain APK-2 (1.22, 0.23and 0.82 μ moles sugar/min/mg protein respectively). The nutrient range of different strain of milky mushroom were found to contain 86.42 to 88.9 per cent moisture, 42.32 to 55.32 per cent total carbohydrates, 18.20 to 24.50 per cent digestible crude protein,3.58 to 4.74 per cent crude fat, 7.91 to 9.84 per cent crude fiber and 8.38 to 9.86 per cent ash content on dry weight basis.
Int.J.Curr.Microbiol.App.Sci (2019) 8(8): 2972-2978 International Journal of Current Microbiology and Applied Sciences ISSN: 2319-7706 Volume Number 08 (2019) Journal homepage: http://www.ijcmas.com Original Research Article https://doi.org/10.20546/ijcmas.2019.808.343 Assessing the Degradation Ability and Nutritional Quality of Different Strains of Milky Mushroom P Venkatesh*, N Revathy and N Kavi Bharathi Department of Plant Pathology, Agricultural College and Research Institute, Madurai, India *Corresponding author ABSTRACT Keywords Degradation ability, Nutritional quality, Milky mushroom, Carbohydrates, Digestible crude protein Article Info Accepted: 22 July 2019 Available Online: 10 August 2019 The experiment was carried Measuring activity of enzyme like exoglucanase, endoglucanase and xylanase and the nutritional quality of different strains of milky mushroom namely viz., CI-1, CI-2, CI-3, MG-4, CI-5 and APK-2 in the study Among the five strains tested, CI-3 recorded maximum activity of endoglucanase, exoglucanase, and endoxylanase (1.50, 1.02, 1.66 μ moles sugar/min/mg protein,) followed by strain APK-2 (1.22, 0.23and 0.82 μ moles sugar/min/mg protein respectively) The nutrient range of different strain of milky mushroom were found to contain 86.42 to 88.9 per cent moisture, 42.32 to 55.32 per cent total carbohydrates, 18.20 to 24.50 per cent digestible crude protein,3.58 to 4.74 per cent crude fat, 7.91 to 9.84 per cent crude fiber and 8.38 to 9.86 per cent ash content on dry weight basis Introduction Milky mushrooms are with great source quality protein, minerals and various vitamins From the age old period of time mushroom were used as a food materials without knowing its medicinal values It is an important food item concerning human health, nutrition and disease prevention Gruen and Wong (1982) indicated that edible mushrooms were highly nutritional and compared favourably with meat, egg and milk food sources Different strains of Calocybe indica ability to adopt wide range of the temperature, pH levels and yield potential depending on the substrates used It is more attractive with excellent shelf-life, grows on several agricultural wastes and on wide range of temperatures (Singh et al., 2015) C indica has great demand in many parts of the country due to its attractive milky white sporocarp, typical wild mushroom taste, fibrous feel of the texture, long shelf This mushroom was first collected in wild form from West Bengal (India) by Purkayastha and Chandra in 1974 Production technology of Calocybe indica has been introduced by Purkayastha and Nayak in 1979 which was improved by Purkayastha and Nayak in 1981.Milky mushroom cultivation has proved its economic strength 2972 Int.J.Curr.Microbiol.App.Sci (2019) 8(8): 2972-2978 and ecological importance for efficient utilization, value-addition and biotransformation of agro-industrial residues (Zervakis and Philippoussis 2000) Commercial mushroom production is yet another efficient and relatively short biological process of food protein recovery from unutilized lignocellulosic materials through enzymatic degrading capabilities of mushroom fungi (Chiu and Moore, 2001) The solution was filtered and stored in dark bottles in refrigerator Materials and Methods Assay Estimation of enzymes The test tubes containing a mixture of 0.5 ml CMC solution and 0.5 ml of appropriately diluted enzyme extract were incubated at 50ºC for 30 minutes in water bath Controls devoid of enzyme extract were also run simultaneously Reducing sugars produced during this reaction were measured by using DNS method ml of DNS was added to each tube and kept in boiling water bath for 15 mins While still hot, ml of sodium potassium tartarate solution was added, the contents were cooled to room temperature followed by addition of ml of distilled water in each test tube The percent light absorbance by the resulting solution was recorded at 575 nm in a spectronic 20 The corresponding enzyme activity was calculated from the standard curve Endoglucanase Celluloytic enzymes namely, Endo-β-D-1, 4glucanase activity was measured by estimation of the reducing sugars released during incubation of the substrate with enzyme extract according to method Mandels et al (1976) The reducing sugars were estimated using DNS reagent (Miller, 1959) Reagents 1) 0.1 M citrate buffer (pH 4.8) A 0.1 M Citric acid B 0.1 M tri sodium citrate 23 ml of A and 27 ml of B and volume made upto 100 ml with distilled water 2) Carboxy Methyl Cellulose solution (CMC) g of Cellulose solution was dissolved in 90 ml of 0.1 M citrate buffer of pH 4.8 and volume was made to 100 ml 4) Sodium potassium tartarate solution (40%) (Rochelle salt) 40 g of sodium potassium tartarate was dissolved in 100 ml distilled water The solution was filtered and stored at room temperature Exoglucanase Cellobiohydrolase activity was determined by the method reported by Mandels and sternberg (1976) Reagents 3) Dinitrosalicylic acid (DNS) solution 10 g of DNS and 0.5g sodium sulfite in 500 ml of 2% NaOH solution already prepared in distilled water were added The solution was allowed to cool, g of phenol was dissolved in it and final volume was made to 1000 ml 1) 0.1 M citrate buffer (pH 4.8) 2) DNS solution 3) Sodium potassium tartarate solution (40%) 4) Filter paper strips (Whatmann no.1, 1x cm) 2973 Int.J.Curr.Microbiol.App.Sci (2019) 8(8): 2972-2978 Assay The test tube containing 0.5 ml enzyme extract and ml of citrate buffer and a filter paper strip were incubated at 50ºC for hour in water bath Controls were run simultaneously The reducing sugars produced during the reaction were estimated by DNS method as described in section 3.16.1 Preparation of standard curve A standard curve for measurement of reducing sugars was prepared under the same conditions Miller (1959), as described above using standard solution of glucose from 0.10.5 mg/ml concentration Enzyme units Enzyme activity of endoglucanase and exoglucanase was expressed as μmoles glucose/min /ml of culture filtrate or /g straw Specific activity was expressed as μ moles glucose /min/mg protein Reducing sugars were measured as xylose equivalents by DNS method as described in 3.16.1 Units of enzyme activity One unit of enzyme activity is expressed as amount of xylanase catalyzed the formation of μmol xylose/min/ml of culture filtrate or /g straw Specific activity was expressed as μmol xylose/min/mg protein Nutritive value of different strains of milky mushroom The various constituents in the sporophores of different strains of milky mushroom(CI-1, CI2, CI-3, CI-5and var APK-2) were analyzed on dry weight basis by the following methods Determination of moisture content Endoxylanase The moisture content of the sporophore was estimated by drying 25 g of fresh sporophore in an oven at 80°C for three consecutive days It was cooled in a desiccator and weighed The moisture content was calculated as, Xylanase (hemicellulase) activity was assayed according to the method of Erikson andBucht (1968) Fresh weight - Dry weight Moisture content (%) = × 100 Fresh weight Reagents Determination of total carbohydrates 1) Xylan solution The anthrone method described by Hedge et al (1962) was followed to determine the total carbohydrates gm of xylan was dissolved in 0.05 M citrate buffer, pH 4.8 and volume made to 100 ml 2) DNS reagent 3) Sodium potassium tartarate solution (40%) Procedure ml of sample of approximately diluted culture was mixed with ml of 1% xylan solution and incubated for 30 minutes at 50ºC One hundred mg of the sample was hydrolysed in a boiling tube by keeping it in a boiling water bath for three hrs with five ml of 2.5 N HC1 and cooled to room temperature This was neutralised with solid sodium carbonate until the effervescence ceased Solution was centrifuged after making the volume upto 100 ml From the 2974 Int.J.Curr.Microbiol.App.Sci (2019) 8(8): 2972-2978 supernatant, 0.5 and ml of aliquots were drawn for analysis Volume was made up to one ml using distilled water and added four ml of anthrone reagent Heated for mins in a boiling water bath and rapidly cooled Green colour obtained was read at 630 nm by using Spectronic-20 Amount of total carbohydrates present was calculated from the standard graph Determination of crude protein The total nitrogen content was estimated by the Micro-kjeldahl method One g of powdered sample was taken in a digestion tube to this 0.05 g of digestion mixture (selenium dioxide, copper sulphate, potassium sulphate (1: 8: 40), ground separately and mixed well) was added followed by 10 ml of conc sulphuric acid The mixture was digested till it turned to colourless solution The digest was then cooled and the volume was made upto 100 ml with distilled water 10 ml of aliquot was taken, distilled and titrated against 0.1 N HC1 by using Micro Kjeltech unit (Vapodest, version 45) A reagent blank with an equal volume of distilled water was run and the titration value was subtracted from the sample titrate value Nitrogen content estimated as N in g kg-1 = (ml HCL – ml blank) Normality × 14.01 Weight (g) The digestible crude protein content was obtained by multiplying the total nitrogen value with the factor 38 (Crisan and Sands, 1978) Determination of crude fat The crude fat in the sporophores was estimated by Soxhlet method (Lees, 1975) In the pre-weighed extraction flask, two g of the dried sporophore was kept in the extraction thimble The thimble was placed in the extractor for the extraction of crude fat using 100 ml of petroleum ether (b p 49 - 60°C) by heating over a water bath After six to eight siphoning, the petroleum ether was evaporated in a water bath The fat content was recorded after cooling in a desiccator Percentage of crude fat was calculated as, (Weight of flask + ether extract) - Weight of flask = ×100 Sample weight Determination of crude fiber The crude fiber content in the sporophore was estimated following the method of De (1965) The residue in the thimble, after extracting crude fat was transferred to a beaker and boiled for 30 mins with 200 ml of 1.25 per cent sulphuric acid The mixture was filtered through a muslin cloth and the residue was washed for free of acid with water The residue was transferred to a beaker containing 200 ml of 1.25 per cent sodium hydroxide and boiled for 30 mins The solution was filtered through muslin cloth and washed with 25 ml of boiling 1.25 per cent sulphuric acid, 350 ml portions of water and 25 ml alcohol Residue was transferred to silica dish and dried fortwo hrs at 130 ± 2˚C Dish was cooled in a desiccator, weighed and ignited at 600+15 °C for 30 mins, cooled and weighed Per cent crude fiber content = 2975 Loss in weight on ignition × 100 Weight of the sample Int.J.Curr.Microbiol.App.Sci (2019) 8(8): 2972-2978 Determination of ash content A quantity of five g of dried and powdered sporophore was ignited in a silica dish for five h at 6000˚ C till a white ash was obtained, cooled and weighed (Raghuramulu et al., 1983) Determination of energy value The energy value of oyster mushroom species was estimated based on the content of crude protein, crude fat and total carbohydrates in the mushroom using the factor 2.62, 37 and 4.2 k cal per gram of each component respectively(Crisan et al., 1978) Energy value (k cal/100 g dry weight) = 62 (% N x 25) +8.37 (% fat) + 4.2 (% total carbohydrates) Results and Discussion Enzyme activity Production of enzyme is of prime importance for efficient degradation of substrates and utilization of nutrients The enzymes such as cellulase and laccase are responsible for degradation of cellulose and lignin content present in the substrates The efficiency of the enzyme production positively correlates with the yield of mushroom Among the five strains tested, CI-3 recorded maximum activity of endoglucanase, exoglucanase, and endoxylanese (1.50, 1.02, 1.66 μmoles sugar/min/mg protein,) followed by strain APK-2 (1.22, 0.23and 0.72 μmoles sugar/min/mg protein) The least level of endoglucanase activity recorded in both the strains CI -2 and CI -5 (0.20 and 0.17 μmoles sugar/min/mg protein) Minimum level of exoglucanase activity was measured in strain CI-5 (0.06 μmoles sugar/min/mg protein) CI1 and CI-5 recorded the low level of endoxylanase activity (0.51 and 0.54 μmoles sugar/min/mg protein) which correlates withRamkumar et al (2011) revealed that CaCO3 (2percent) amended Czapeck’s Dox liquid medium recorded the high level of lignocellulolytic enzyme production viz., exoβ-1,4 glucanase 2.31 and endo β-1,4 glucanase 1.59, β glucosidase (1.79), xylanase ( 1.94), laccase polyphenol oxidase(0.82) in Lentinuseddodes (1.85) and Bhupathi et al (2017)recorded maximum level of xylanase at all the seven stages of mushroom growth followed by lipoxygenase activity Maximum activity of xylanase was recorded in the pileus of APK-2 variety and CBE-TNAU-1523 wild strain (3.514 mols/min/g and 3.55 moles/ min/g respectively) when compare to stipe(Table 1) Table.1 In vitro activity of endoglucanase, exoglucanase and xylanase production of different strains of milky mushroom S.No Strains C.indica- CI-1 C indica- CI -2 C indica-CI-3 C indica- CI-5 C indica -APK-2 CD (P=0.05) Cellulase(μmoles glucose /ml) Endoglucanase Exoglucanase 1.09 0.19 0.20 0.14 1.50 1.02 0.17 0.06 1.22 0.23 0.05 0.01 2976 Xylanase Endoxylanase(1μmol xylose/ml) 0.72 0.51 1.66 0.54 0.82 0.04 Int.J.Curr.Microbiol.App.Sci (2019) 8(8): 2972-2978 Table.2 Proximate composition of milky mushroom (percent dry weight basis) S.No Strains of milky mushroom C indica-CI-1 C indica-CI -2 C indica-CI-3 C indica - CI-5 C indica-var APK-2 CD(P=0.05) Moisture content 86.7 88.9 86.4 88.0 87.5 TotalCarbohy drate 55.32 50.12 42.32 48.50 47.08 Crude protein 18.20 19.39 24.50 19.85 20.2 Crude fat 3.82 4.01 3.58 4.74 3.62 Crude fiber 8.65 7.91 9.84 9.32 8.54 Ash 9.2 8.38 8.64 9.86 9.52 4.1 1.83 1.08 0.17 0.32 0.50 CalorieVal ue (k/100g) 328.46 310.26 229.5 305.98 301.7 content of 0.12 mg/ g, 0.09 mg/ g and 0.06 mg/ g respectively (Table 2) Proximate composition of milky mushroom Mushrooms are considered as a one of world’s greatest untapped resources of nutritious and palatable food (Subramanian et al (2015) Pani et al (2012) revealed that milky mushroom are rich in various nutrient source like protein, fiber, mineral, carbohydrate and more amount of essential amino acids The results registered that different strains of milky mushroom were found to contain 86.42 to 88.9 per cent moisture, 42.32 to 55.32 per cent total carbohydrates, 18.20 to 24.50 per cent digestible crude protein,3.58 to 4.74 per cent crude fat, 7.91 to 9.84 per cent crude fiber and 8.38 to 9.86 per cent ash content on dry weight basis They possessed an energy value of 229.5 to 311.34 k cal/100 g Alamet al (2008) suggested that the 100 g of dried mushroom consists 24g of protein, 4.5g of lipid, 12.9g of fiber, 13.1g of ash, 48.5 % of carbohydrate and 87 % of moisture level Doshiet al (1988) reported that mature sporophore of Calocybeindicahad soluble sugars (4%), starch (2.9%) and ash (7.4%) Dhakad et al (2017) conducted an experiment on five different strains of milky mushroom to estimate the nutrient status and founded that highest level (0.14mg/g of fresh mushroom) of protein content in the strain CI-8,other strain CI-15,CI-13 and CI-14 had the protein References Alam, N., Amin, R., Khan, A., Ara, I., Shim, M J., Lee, M W., & Lee, T S (2008) Nutritional analysis of cultivated mushrooms in Bangladesh-Pleurotus ostreatus Pleurotus sajor-caju, Pleurotus florida and Calocybe indica Mycobiology 36(4), 228-232 Bhupathi, P., Krishnamoorthy, A S., & Uthandi, S (2017) Profiling of morphogenesis related enzymes of milky mushroom Calocybe indica (P & C) Journal of Pharmacognosy and Phytochemistr 6(5), 2537-2543 Crisan, E., & Sands, A (1978) Nutritional value: Academic Press, New York Chiu SW, Moore DA.(2001) Threats to biodiversity caused by traditional mushroom 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Sing Emirates Journal of Food and Agriculture, 23(1), 71 Subramanian, K., & Shanmugasundaram, K (2015) Optimization of casing process for enhanced bioefficiency of Calocybe indica, an indigenous tropical edible mushroom International Journal of Recent Scientific Research, 6(2), 25942598 Zervakis G, Philippoussis A Management of agro-industrial wastes through the cultivation of edible mushrooms In Proceedings of IV European Waste Forum ‘Innovation in waste management’, CIPA, Milan, 2000 How to cite this article: Venkatesh, P., N Revathy and Kavi Bharathi, N 2019 Assessing the Degradation Ability and Nutritional Quality of Different Strains of Milky Mushroom Int.J.Curr.Microbiol.App.Sci 8(08): 2972-2978 doi: https://doi.org/10.20546/ijcmas.2019.808.343 2978 ... protein Nutritive value of different strains of milky mushroom The various constituents in the sporophores of different strains of milky mushroom( CI-1, CI2, CI-3, CI- 5and var APK-2) were analyzed... this article: Venkatesh, P., N Revathy and Kavi Bharathi, N 2019 Assessing the Degradation Ability and Nutritional Quality of Different Strains of Milky Mushroom Int.J.Curr.Microbiol.App.Sci 8(08):... responsible for degradation of cellulose and lignin content present in the substrates The efficiency of the enzyme production positively correlates with the yield of mushroom Among the five strains tested,